The functional domains of the HIV-1 Gag matrix (MA) protein were identified and characterized by introducing more than 70 single and double amino acid substitutions throughout the MA coding region. The effects of these mutations on Gag expression and processing, virus particle assembly and production, virus infectivity in a range of cell types, and envelope (Env) glycoprotein incorporation into virions, were determined. Several classes of mutations were identified, including those that: I) blocked virus assembly, ii) redirected particle assembly from the plasma membrane to cytoplasmic compartments, iii) interfered with virus entry, or iv) blocked Env incorporation into virions without affecting virus assembly and release. During the course of this analysis, viral revertants of all mutant classes were obtained. Second-site changes were identified that could compensate, both biologically and biochemically, for the effect of the original mutation. For example, a Val->Ile change at residue 34 compensated for two position 12 mutations which blocked Env incorporation into virions, a Gln->Lys substitution at residue 27 (in the MA highly basic domain) reversed the phenotype of a position 86 mutation which redirected virus assembly to the cytoplasm, and substitutions at residues 10, 69, or 97 restored virus particle production in the context of a mutation at MA amino acid 6. These results have important implications for understanding MA structure/function relationships and delineating the role of MA in the HIV-1 life cycle. The enzymes (reverse transcriptase, integrase, and protease) associated with infectious HIV-1 particles are derived from the 160 kDa Gag-Pol precursor protein, which is thought to be incorporated into nascent virions via its interaction with the 55 kDa Gag precursor (p55). The region within the Gag-Pol precursor directing its incorporation into nascent virus particles was mapped by constructing chimeric HIV-1/MuLV Gag-Pol proteins and monitoring their interaction with wild type HIV p55. A defined segment within the CA domain was found to be necessary and sufficient for incorporation of the HIV-1 or heterologous Gag-Pol precursors into progeny virions.